Method and kit for identifying or characterizing polypeptides

ABSTRACT

Polypeptides which have been separated by gel electrophoresis can be identified or characterized by a procedure which has two main stages. In the first stage the gel is digested with a polypeptide-cleaving agent such as an enzyme. This produces mainly large fragments which, in the second stage are electroblotted through a hydrophilic membrane on which is immobilized another polypeptide-cleaving reagent such as an enzyme onto a hydrophobic member, typically a membrane, e.g. of PVDF. The resulting fragments, usually peptides, are identified, preferably by MALDI-TOF MS, or a property may be determined, e.g. by interaction with an antibody.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the identification or characterisation of oneor more polypeptides which have been isolated on a gel, typically frompolyacrylamide gel electrophoresis (PAGE) and to a kit for use in themethod. It is especially useful in proteomics (the large scaleidentification and characterisation of proteins).

2. Description of the Related Art

In proteomics, massively parallel protein identification andcharacterisation techniques are required. The identification of proteinsor other polypeptides merely by PAGE, even using two-dimensional gels(2D-PAGE), is laborious and often uncertain. Many different methods havebeen developed to identify and partially characterise proteins fromcomplex biological samples. Some of them use Matrix Assisted LaserDesorption/Ionization-Time of Flight Mass Spectrometry (MMI-TOF MS)techniques to analyse peptide “fingerprints” produced by fragmenting theproteins with enzymes. Several software programs have been developed tocompare mass spectra of the peptides obtained from MALDI-TOF MSexperiments with theoretical spectra from proteins. The subject has beenreviewed by M. Kussmann and P. Roepstorff, Spectroscopy 1998, 14, 1-27.These authors noted three ways in which proteins separated by gelelectrophoresis could be digested with enzymes to yield fragmentpeptides:

1. The digestion can be carried out in a plug of excised gel and thepeptides recovered by elution. This is the authors' own preference.

2. The protein can be first electroleluted from an excised gel plug andthen digested in solution.

3. The protein can be electroblotted onto a membrane and subsequentlydigested on the membrane.

These types of processes are not practical for the sequencing ofpolypeptides which have been run on the same gel, since the cutting outof the polypeptide bands from the gel has to be done sequentially andthe plugs thus obtained placed in tubes for further analysis. Also,losses occur when the polypeptides adhere to the walls of the tube.

Two of the present inventors have experimented with a different method,which they have termed one-step digestion transfer (OSDT). See U.S.patent application Ser. No. 09/107 991 filed Jun. 30, 1998 andcorresponding Canadian Patent Application No. 2 244 947 filed Sep. 24,1998 entitled “Methods of identifying polypeptides”, the disclosure ofwhich is herein incorporated by reference. They have found that theproteins or other polypeptides separated on a gel can be cleaved intofragments, for example by digestion with an enzyme, and that thesefragments are presented very satisfactorily for analysis, especially byMALDI-TOF NS, if the cleaving reagent is immobilised on a hydrophilicmembrane and interposed as the “filling” in a blotting “sandwich”between the separation gel as one “slice” of the sandwich and ahydrophobic collection member, exemplified as a conventionalpolyvinylidene fluoride (PVDF) membrane, as the other “slice” of thesandwich. In this way, the fragments are collected on the hydrophobicmember and can then be formulated in an appropriate way for theMALDI-TOF MS. It is only necessary that the transblotting is carried outso that the proteins have a long enough residence period in theproximity of the immobilised cleaving reagent to ensure that areasonable amount of the fragments is produced, but, of course, not solong as to allow undesired diffusion. With electroblotting, i.e.blotting assisted by an electric field, this is easily achievable byvarying appropriately the current used in the electroblotting, e.g. bypulsing the current or using a unsymmetrical alternating current.Further, when an enzyme is used as the cleaving agent and when theenzyme is immobilised securely on a hydrophilic membrane, especially bycovalent bonding to the solid phase, autodigestion (cleavage of theenzyme by itself) is inhibited.

The OSDT method gives good results for many proteins, but very stronglybasic proteins such as lysozyme are not easily transferred under theconditions which are optimal for use of the preferred enzyme, trypsin.Trypsin gives best digestion in a buffer of pH about 8.4. Also, the OSDTmethod does not give good digestion of very high molecular weightproteins.

SUMMARY OF THE INVENTION

The present invention Is an improvement to the OSDT method and is basedin part on the discovery of another technology which the inventors havetermed “in full gel digestion” (IFG). This procedure involvesdehydrating the gel and then rehydrating it, adding to gel apolypeptide-cleaving reagent such as an enzyme, e.g. in the rehydrationbuffer. After the IFG, the digested proteins are then electroblotted ina conventional way. One drawback of this technique is the loss of lowmolecular weight proteins (those of m.w. less than 40 kDa) by diffusionduring the in-gel digestions.

It has now been found that by combining the IFG procedure, optionallymodified, with OSDT, satisfactory digestion of the proteins (or otherpolypeptides), accompanied by improved identification, can be achievedfor polypeptides having a wide range of molecular weights. Moreover,high molecular weight proteins can be satisfactorily immunoblotted toyield fragments which can be identified as epitopes.

In a preferred “combined procedure”, the gel is dehydrated and at leastpartially, preferably only partially, rehydrated with a buffercontaining the polypeptide-cleaving reagent, IFG is performed and thisis then followed by OSDT.

In one aspect the invention provides a method of identifying orcharacterising polypeptides which have been isolated on a gel byelectrophoresis, comprising:

a) providing a gel on which at least one polypeptide has been isolated;

b) incorporating a first polypeptide-cleaving reagent in the gel(preferably by dehydrating the gel and at least partially rehydrating itwith a buffer containing the polypeptide-cleaving reagent);

c) providing adjacent to the gel at least one hydrophilic membrane onwhich is immobilised at least one second polypeptide-cleaving reagent;

d) providing a hydrophobic collection member (preferably a membrane)suitable for receiving thereon fragments of polypeptide transferredthereto from the gel by transblotting, preferably by electroblotting,said hydrophobic member being positioned beyond the hydrophilic membranein a direction of movement of the fragments of polypeptide;

e) transblotting the polypoptide or polypeptides from the full gel, onwhich the polypeptide or polypeptides were isolated, through thehydrophilic membrane or membranes, under conditions effective to causeit or them to be cleaved into fragments by the secondpolypeptide-cleaving reagent, to the hydrophobic member; and

f) identifying or characterising the fragments collected on thehydrophobic collection layer.

Preferably the method further comprises

g) identifying or characterising the polypeptide from which thefragments were derived.

The invention also includes a kit for use in the method of theinvention, said kit comprising:

a) a first polypeptide-cleaving reagent suitable for incorporating in anelectrophoretic gel;

b) at least one hydrophilic membrane suitable for use in transblottingof polypeptides separated on an electrophoretic gel, the membrane havingat least one second polypeptide-cleaving reagent imobilised thereon; and

c) a hydrophobic collection member suitable for receiving thereonfragments of the separated polypeptides transferred thereto bytransblotting.

Elements b) and c) may be provided as separate components, e.g. inseparate containers, or as a pre-formed assembly.

The term “cleaving a polypeptide”, as used herein, refers to any step inwhich a group, residue or any chain of groups or residues is split offfrom the remainder of the molecule. It includes cleavage in the mainchain of amino acids or in a side-chain or of any terminal or side-chaingroup or residue, e.g. removal of a C-terminal amino acid bycarboxypeptidase, N-terminal amino group by an aminopeptidase or aglycosyl side-chain by a glycosidase is included.

The method of the invention requires digestion in the full gel. That is,the method does not include cutting pieces from the gel and digestingthe cut pieces in an enzyme.

Reference above to the gel having been dehydrated covers allowing it tobecome dehydrated merely by standing in air at ambient temperature ortaking deliberate steps to dehydrate it. The term “dehydration” includescomplete, substantially complete or partial removal of water from thegel.

The term “collection member” as used herein has a broad meaning, sincethis is not in itself critical to the invention. Considered inisolation, it may be, for example, a self-supporting membrane, film, orplate, or it may be non-self-supporting, e.g. a hydrophobic layersupported on a substrate, e.g. as a coating. It will normally be porousto the blotting buffer, to enable current to be carried to or from theelectrode, but may alternatively be the electrode or in directelectrical communication with it.

The term “transblotting”, as used herein, covers any operation oftransferring the polypeptide fragments to another surface, which, inthis invention, is a hydrophobic collection ember. It includes a processof transfer by capillary action or by electroblotting. In thisspecification, “transblotting” can be part of any blotting procedureapplicable to polypeptides, including, for example immunoblotting.

The term “identifying” as used herein is not synonymous with determiningthe sequence and includes partially identifying the polypeptide.Further, it includes making a tentative identification based on the mostprobable of a mall number of possibilities.

The term “kit” as used herein includes combinations of the identifiedcomponents in separate containers and also an assembly of thehydrophilic membrane(s) and hydrophobic collection layer ready for use.The kit may further include, in separate containers, other reagentsuseful in the method of the invention, e.g. a buffer for rehydrating thegel, a blotting buffer, reagent(s) which assist in the reaction of theenzyme with the polypeptide fragment and so on.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are schematic views of two kinds of blotting “sandwich”which can be used in the invention;

FIG. 3 is a plot of applied voltage against time, showing the productionof an alternating voltage for use in electroblotting in the method ofthe invention;

FIGS. 4A-4D and 5A-5D show stained polypeptide bands present on thecollection membrane to which the proteins and protein fragments havebeen transferred, respectively, for control (4A, 5A), IFG digestion (4B,5B), OSDT (4C, 5C) and the combined method of the invention (4D, 5D);and

FIGS. 6A-6C and 7A-7C show the MALDI-TOF MS spectra obtained from myosinand chicken lysozyme respectively, for IFG (6A, 7A), OSDT (6B, 7B) andthe combined method of the invention (6C, 7C).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to identifying polypeptide(s) which have alreadybeen isolated on a gel by gel electrophoresis. The nature of thepolypeptide(s) to be identified is not critical. They can be, forexample, naturally occurring proteins, proteins made by recombinant DNAtechnology, polypeptide(s) made by peptide synthesis or by expression ofrecombinant DNA. For brevity, the invention will be describedhereinafter with reference to proteins. The extrapolation to otherpolypeptide(s) will be taken as understood and incorporated throughoutthe following description.

The kind of gel on which the proteins have been isolated is notcritical, but will usually be a polyacrylamide gel. Any of theconventional gels and separation conditions may have been employed,including reducing conditions. They may be one-dimensional ortwo-dimensional gels. (In 2D gels, proteins etc. are separated in onedimension by their charge and in the other dimension by their molecularmass).

The invention is normally to be applied to multiple proteins co-presenton the same gel, for example from 3 to 3000, more usually 30 to 3000 andpreferably 50 to 1500, proteins. This includes proteins present atdifferent molecular weight separations on a 1D gel or at similarmolecular weight separations, but present in parallel lanes or tracks onthe 1D gel, as well as those separated by 2D gel electrophoresis.However, the invention also applies to a gel on which a singlepolypeptide is required to be identified or characterised. Inparticular, it is useful in relation to immunoblotting of proteins ofhigh molecular weight, e.g. 150-250 kDa, in order to split them intofragments in which epitopes can be recognised by immunoblotting.

The first stage of the method of the invention comprises digestion ofthe proteins within the same gel as that on which they were separated.Within this stage, there are preferably three main operations. The firstoperation is to dehydrate the gel. The gel may be fully or partiallydehydrated depending on the amount of protein digestion required. Thegreater the dehydration the greater the capacity of the gel subsequentlyto absorb a solution containing the first polypeptide-cleaving reagentand therefore the greater the digestion. Dehydration may be complete,substantially complete, e.g. by removal of 90-99% by weight of theoriginal water content, or, more preferably, partial, e.g. by removal of25-90%, preferably 40-70%, on the same basis. Of course, it is moretroublesome to rehydrate a completely dried-out gel. The method ofdehydration is not critical. Air-drying at ambient temperature, say15-25° C. is preferred. Mere standing of the gel at 4-10° C. under lowpressure is another method. Air-drying, followed by standing, is afurther method.

The next operations are rehydration and the incorporation of the firstpolypeptide-cleaving reagent in the rehydration buffer. Thepolypeptide-cleaving reagent is preferably an enzyme such as trypsin orLys-C, but a chemical cleaving agent such as CNBr could alternatively beused. Examples of other enzymes and chemical cleaving reagents are givenlater, in connection with the discussion of the secondpolypeptide-cleaving reagent. Hereinafter enzymes will be referred to,the extrapolation to other polypeptide-cleaving reagents being taken asunderstood and incorporated mutatis mutandis in the followingdescription. The first enzyme may be the same as or different from thesecond enzyme(s) used in the transblotting step. Any of the enzymesdescribed below for use in transblotting can be used in the in full geldigestion.

In principle, it is immaterial at what stage the first enzyme isintroduced into the gel and whether it is present in the gel in a freeor immobilised form. However, if present in the gel from the start ofelectrophoresis, it would normally upset the pattern of proteinseparation, unless the enzyme or conditions of running the gel were sochosen as to make it inactive while the gel is being run. This could beachieved by adding a reversible inhibitor, of the enzyme to the runningbuffer. For example, if the enzyme is trypsin a reversible inhibitorsuch as benzamidine would be suitable. Conditions are e.g. as describedin S. L. Jeffcoate et al., J. Clin. Endocrinal. Metab. 1974 38, 155-157.Then, after the gel has been run, resulting in separation of proteins,it is washed as part of the rehydration step, resulting in the removalof inhibitor and therefore re-activation of the enzyme.

The enzyme is normally added to the gel after isolation of the proteins.It is added as a solution or fine suspension which will penetrate thegel and be absorbed well by the gel solids. In principle, the enzymecould be added before any deliberate step of rehydration or even afterrehydration has been effected, e.g. in a concentrated solution. However,in practice, such techniques are unlikely to give the best digestion.Normally, the enzyme will be present in the rehydration solution, whichis buffered. Preferably the gel is incubated with the rehydration buffercontaining the enzyme, for example for 30 minutes at 35° C. The time andtemperature can be varied, according to the size of fragments desired.Rehydration may be partial, complete or even to an extent in which thegel contains more water than when the proteins were run on it. Anyextent of rehydration appropriate to allow transblotting is permissible.In practice, it is convenient to treat the gel with excess rehydrationsolution and to remove the excess. Otherwise, undesired proteindiffusion or excessive swelling of the gel can occur.

At this stage the gel contains peptides with a high number of missedcleavages, from partially digested proteins. The fragments of highmolecular weight and basic proteins are more easily extractable from thegel by electroblotting, compared with the full molecule.

The second stage of the method of the invention comprises transblotting,preferably electroblotting. Normally, the electroblotting takes placeoverall in the direction cathode to anode, as the proteins arenegatively charged. Depending on the pH of the electroblotting bufferused, positively and negatively charged fragments could be obtained andmigrate in opposite directions, towards the cathode and anoderespectively. FIGS. 1 and 2 of the drawings exemplify some sandwichesfor the electroblotting. FIG. 1 shows an experimental arrangement inwhich a cathodic collection layer, which is preferably a conventionalPVDF membrane, was provided, just to show that under these conditions noproteins migrated to this membrane, despite the alternating fieldapplied (thus reversing the electrodes). It will be understood thatunder different pH conditions, some fragments could be produced at thecathodic collection layer. Thus, the invention includes the possibilityof providing anodic and cathodic collection layers, with hydrophilicmembranes interposed between each of them and the separation gel layer.In FIG. 1 there is a single hydrophilic membrane, which is preferably amodified PVDF membrane, having an appropriate protein-cleaving reagent,normally a protease enzyme, for example trypsin, immobilised on it,interposed between the gel layer and an anodic collection layer, mostconveniently a conventional PVDF membrane, on which the proteinfragments are collected. In FIG. 2 there is no cathodic collectionlayer, but there are two consecutive hydrophilic membranes, preferablymodified PVDF membranes, each with trypsin immobilised thereon, placedbetween the gel layer and the anodic collection layer, which, again, ispreferably a conventional PVDF membrane.

In more detail, the anode and cathode are separated from the rest of thesandwich by an absorptive layer which soaks up the blotting liquid,while maintaining the liquid in electrical contact with the electrodes,and is conveniently a filter paper. The kinds of electrodes andabsorptive layers used in arrangement are not critical and can be anyconventionally used in electroblotting.

The anodic collection layer (and cathodic collection layer if used) arealso not critical and thus can be any conventional hydrophobic membraneused in electroblotting, such as PVDF (conventional or positivelycharged) nylon or nitrocellulose, for example.

The “filling” of the sandwich can take the form of one or more membranes(defined as above) sufficiently hydrophilic in character that theproteins and fragments thereof do not tend to stick thereon. Thismembrane can be formed from any thin member which is porous to theelectroblotting liquid and capable of immobilising thepolypeptide-cleaving reagent thereon, whether on the surface thereof ofwithin interstices or microcavities therein accessible to theelectroblotting liquid (and therefore to the polypeptide to be cleaved).It will typically be from 100 to 600 μm thick. Usually the number ofsuch membranes will be from 1 to 3. With conventional thicknesses ofmembrane, e.g. 130 to 150. Am as in the preferred “Immobilon AV” PVDFmembrane, 2 membranes will frequently be used. They are best placeddirectly mutually adjacent, i.e. one on top of another. Preferably, thesecond polypeptide cleaving reagent is bonded to the hydrophilicmembrane covalently. For this purpose, the hydrophilic membrane(s) arepreferably provided with “active carbonyl” or carboxylic acid groups orderivatives thereof reactive with amino groups present in the cleavingreagent, e.g. an enzyme. “Active carbonyl”-modified or carboxyl-modifiedPVDF membranes are especially preferred.

Since it would be difficult to react all the active groups present onthe surface of a membrane with an enzyme, and since it is undesirable toallow the polypeptides to react with these free active groups, theresidual active groups (which would otherwise be free) are preferablycapped before the membrane is used, e.g. with ethanolamine, thusproviding terminations such as —CO—NH—CH₂—CH₂—OH, which are relativelyhydrophilic. Other hydrophilic capping groups will suggest themselves tothose skilled in the art.

Alternatively, PVDF membranes or glass fibre paper can be functionalisedby isothiocyanate, which allows reaction with the N-terminal aminogroups and/or the ε-amino groups of lysine residues in the enzymes. Forthis purpose, the PVDF membranes are pre-treated with NaOH to provide acarbon-carbon ethylenic double bond in the polymer chain, by eliminationof a molecule of HP. The pre-treated PVDF membranes are then reactedunder basic conditions with a dinucleophile such as ethylenediamine, 1,10-diaminodecane or 2-aminoethanethiol, whereby hydrogen atoms in thepolymer are substituted by —X—(CH₂)_(n)—NH₂ groups, wherein —X— is —S—or —NH— and n is 2 or 10. This polymer, having amine-terminatedside-chains, is then reacted with 1,4-phenylenediisothiocyanate (DITC)or 3,5-dichloro-1,4-phenylenediisocyanate (DCDITC) to give the requiredisothiocyanate-terminated side-chains in good yield. DITC-reacted glassfibre sheets provide another form of membrane, see R. H. Aebersold etal., J. Biol. Chem. 1986, 291, 4229-4338.

Another form of hydrophilic membrane is PVDF functionalised by arylaminegroups, which react with a carboxylic acid side-chain or the carboxylterminus of the enzyme, preferably in the presence of a carbodiimidesuch as 1-(3-dimethylaminopropyl)-3-ethylcarbodimide.

Another form of hydrophilic membrane which can be used as the sandwichfilling is a thin film or coating of agarose gel. The H-terminal aminoand/or ε-amino groups (according to the selectivity of the reaction) oflysine residues in the enzyme are treated to obtain aminoxy groups,which react with aldehyde groups produced by mild oxidation of theagarose gel, thus bonding the enzyme covalently to the agaraose.

A further kind of hydrophilic membrane may comprise one or more thinfilms or coatings of polyacrylamide gel, similar in thickness to thatused in immobilised pH gradient electrophoresis (IPG), but which hasbeen trypsinated. This can be done by reacting trypsin with acryloylchloride to form an N-acryloyltrypsin, which is then copolymerised withacrylamide in the preparation of an acrylamide copolymer gel.

Yet another form of hydrophilic Membrane is a glass fibre paper whichhas been modified to replace amino groups by groups containing a diazolinkage, e.g. 4-N,N-dimethylaminoazobenzene-4′-isocyanate groups. Thereactions required for this purpose have been described by J. Y. Changet al., FEBS Letters 1977, 84, 187-190.

The cleaving reagent immobilised on the membrane is normally andpreferably immobilised by covalent bonding. However, other forms ofimmobilisation are not excluded from use in this invention, so long asthe enzyme does not become sufficiently free in solution in theelectroblotting liquid as to undergo autodigestion. (It will beunderstood that the presence of autodigested enzyme fragments couldupset the analysis of the fragments from the protein to be analysed).Thus, for example, the enzyme could be physically trapped within thepores of a porous sheet of hydrophilic polymer. Alternatively, themembrane could have an enzyme immobilised thereon by means comprising(consisting of or including) affinity bonding. Thus, the enzyme could becovalently attached to avidin or streptavidin and the resultantconjugate attached to a biotinylated membrane by affinity bondingbetween avidin/streptavidin and biotin. Alternatively, avidin orstreptavidin could be attached to the membrane and the enzyme could bereacted to provide biotinyl terminations for reaction with a membrane towhich avidin or streptavidin has been attached.

Preferably either or both polypeptide-cleaving reagents comprise anenzyme. If both comprise an enzyme, it may be the same or different.Most preferably and usually, the enzyme cleaves the main chain of thepolypeptide (i.e. is an endopeptidase or endoproteinase), especiallytrypsin. Trypsin cuts proteins at the C-terminal end of many lysines andarginines. Other less specific endoproteases, e.g. pepsin or such aschymotrypsin are usable, as are highly specific enzymes such as Lys-C,Arg-C or Glu-C. For phosphoproteins, a phosphorylase is useful. Eitheror both enzymes can be an exo-enzyme which splits off a side-chain ofthe protein or acts at the terminus. More than one enzyme can beincorporated in the gel. More than one enzyme can be immobilised on themembrane. For example, it may be helpful to split off one or more sidechains of the polypeptide, e.g. using a carboxypeptidase oraminopeptidase, in conjunction with an endoproteinase. CarboxypeptidaseY is one particularly useful such enzyme.

To investigate the presence of side-chains in proteins, such asglucosyl, N-acetyl-O-glucosaminyl and sialyl, enzymes which will cleavethose chains, such as glucosidase, N-acetylglucosaminidase andneuraminidase, respectively, are useful in the invention.

The following chart indicates the various possibilities for types ofenzymes which may be used in combination:

IFG OSDT Endopeptidase, e.g. trypsin Endopeptidase, e.g. trypsinEndopeptidase, e.g. trypsin Exo-enzyme, e.g. glucosidase, phosphorylaseExo-enzyme, e.g. Endopeptidase, e.g. trypsin glucosidase, phosphorylaseExo-enzyme, e.g. Exo-enzyme, e.g. glucosidase, phosphorylaseglucosidase, phosphorylase

The cleaving reagents are not confined to enzymes. Either or both can bea chemical reagent, for example cyanogen broumide, 2-iodosobenzoic acidor a derivative thereof or hydroxylamine. Such reagents are described byE. A. Carrey, “Peptide Mapping” in “Protein Structure: A PracticalApproach”, ed. T. E. Creighton, IRL Press, 1989, pages 117-121. For useas the second polypeptide-cleaving agent, the chemical reagents aresuitably immobilised on the hydrophobic member. Thus, cyanogen bromidecan be physically immobilised by entrapment within pores of thehydrophilic membrane. 2-iodosobenzoic acid can be derivatised,preferably at the COOH group, e.g. with an alkylenediamine, especially1,6-hexanediamine, leaving a free amino group which is then reacted withfunctional groups on the membrane, such as active carboxyl groupsmentioned above.

It will be appreciated that different cleaving reagents will havedifferent specificities and in certain cases the absence of smallfragments, indicating the absence of cleavage, may be a useful resultfor identification or characterization.

The electrical current applied in the electroblotting is preferably nota direct, continuous current, but either pulsed, i.e. a direct currentwith intervals in which no current is passed, or an alternating current.It may be unbiased or biased in the cathode to anode direction, i.e.mainly a cathode to anode current, but with intervals in which currentis passed in the opposite direction. Variations on these regimes arepossible within the general spirit of the idea of performing a slowerthan normal electroblotting, allowing sufficient time for the cleavageto take place on the hydrophilic membrane(s), while avoiding such a slowjourney of the protein fragments from the separation gel to thecollection membrane that lateral diffusion occurs, causing loss ofresolution.

The electroblotting liquid is not critical to obtaining some form ofprotein fragmentation and hence a useful identification. It is normallybuffered and can be any conventional buffer for this purpose, such asTris/glycine with methanol or 3-(cyclohexylamino)-1-propanesulfonic acid(CAPS) with methanol. The direction of migration of the fragmentsdepends essentially on the pH of the buffer. For most purposes analkaline buffer will be appropriate, since many enzymes function best atalkaline pH. Particularly, in the case of trypsin, the buffer willpreferably have a pH from 8.0 to 9.0 and most especially 8.2 to 8.6,with a half Towbin's buffer of pH about 8.4 being considered optimal.Some other enzymes, such as endoproteinase V8, require an acidic pH.Under such conditions, the fragments will migrate to the cathode.

It has been found desirable to incorporate a avail amount of aconventional detergent such as SDS in the buffer, to produce micelles(negatively charged aggregates).

The protein fragments, whether they are peptides derived from the mainchain of the protein or are residues of a side-chain, are collected onthe collection layer. They are then preferably analysed by aspectroscopic method based on matrix-assisted laserdesorption/ionisation (MALDI) or electrospray ionisation (ESI). Thepreferred procedure is MALDI with time of flight (TOF) analysis, knownas MALDI-TOF MS. This involves forming a matrix on the membrane, e.g. asdescribed in the literature, with an agent which absorbs the incidentlight strongly at the particular wavelength employed. The sample isexcited by UV, or IR laser light into the vapour phase in the MALDI massspectrometer. Ions are generated by the vaporisation and form an ionplume. The ions are accelerated in an electric field and separatedaccording to their time of travel along a given distance, giving amass/charge (m/z) reading which is very accurate and sensitive. MALDIspectrometers are commercially available from PerSeptive Biosystems,Inc. (Frazingham, Mass., USA) and are described in the literature, e.g.M. Kussmann and P. Roepstorff, cited above.

In this invention, the above method is applied to the scanning of thefragments of many proteins at once. Thus, many proteins can be runsimultaneously on a polyacrylamide gel, subjected to the method of theinvention to produce an array of spots on the collecting membrane andthe array analysed as follows. After the PVDF membrane or otherhydrophobic collection layer has been stained, a piece of it will be cutand fixed on the MALDI-MS sample plate, e.g. with silicon grease. Anorganic matrix-foming reagent is added to the membrane on the sampleplate and the sample is then air-dried to form the matrix. The sampleplate is inserted in the MALDI-MS spectrometer. An automated movement ofthe sample plate from a first to a second position and subsequentpositions, to align the laser with individual spots in the array, isarranged by computer program. At each position a MALDI-MS spectrum isgenerated, the spectral information collected in digital form and thedata downloaded to the ExPASy database research program (PeptIdent).

It is then relatively simple to provide automated output of the resultsby using the ExPASy server, as at present used for MIDI-TOF MS and togenerate the data in a form handleable by computers.

It will be evident, therefore, that the present invention has hugepotential for the automated identification and/or partialcharacterisation of proteins, e.g. in proteomics research. In effect,the invention provides in this preferred embodiment a “molecularscanner” for this purpose.

Other techniques for improving the mass accuracy and sensitivity of theMALDI-TOP MS can be used to analyse the fragments of protein obtained onthe collection membrane. These include the use of delayed ionextraction, energy reflectors and ion-trap modules. In addition, postsource decay and MS—MS analysis are useful to provide further structuralanalysis. With ESI, the sample is in the liquid phase and the analysiscan be by ion-trap, TOF, single quadrupole or multi-quadrupole massspectrometers. The use of such devices (other than a single quadrupole)allows MS—MS or MS^(n) analysis to be performed.

Still other methods of analysis comprise immunoblotting using monoclonalor polyclonal antibodies and phospho-imaging.

Optionally, other components may be included in the kit, especially anyone or more of the following: matrix-forming reagent for MALDI-TOF,rehydration buffer, electroblotting buffer, collection layer(s), e.g.cationic membranes and PAGE materials. In the case of immunoblotting,the kit may further comprise one or more antibodies, especially a rangeof monoclonal antibodies. Kits as defined above, but further comprisingany one, two or more of the above optional components are herebyspecifically declared to be within the invention. All components of thekit may be supplied in separate containers, but packaged overall as akit.

The following Examples illustrate the invention. The words “Immobilon”,“Trans-Blot”, “Trizma”, “Tween” and “Voyager” are Trade Marks and/orRegistered Trade Marks.

EXAMPLES Materials and Methods

Chemicals. “Immobilon” type AV (IAV) membranes were purchased fromMillipore (Bedford, Mass., USA). Acrylogel-PIP 2.6% C solution waspurchased from BDH (Poole, England). Broad range SDS-PAGE standard PVDFmembranes were purchased from Bio-Rad (Richmond, Calif., USA).Trifluoroacetic acid (TFA), “Trizma base”(Tris),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and trypsin (type IXfrom porcine pancreas, dialysed and lyophilised) were purchased fromSigma (St. Louis, Mo., USA). Acetonitrile (preparative HPLC grade),calcium chloride, ethanolamine, glycine and α-tosyl-L-arginine methylester (TAME) were purchased from Fluka (Buch, Switzerland).

12.5%, 2.6% C linear gel (home made). To produce a 12.5% T, 2.6% Clinear polyacrylamide gel, crosslinked with PIP(“PIP”=N,N′-diacryloylpiperazine), 8 ml of Acrylogel-PIP 2.6% C stocksolution were mixed with 5 ml of Tris-HCl 1.5 M pH 8.8 and 6.6 ml ofdeionized water The polymerisation of the gel was induced with 20 μl ofTEMED and 100 μl of APS (10% w/v). The solution was degassed and loadedinto a Bio-Rad mini-2D gel support. To preserve the gel from theatmosphere, 0.5 ml of water-saturated sec-butanol was added on the topof the gel. After 30 minutes, the gel was washed for subsequent loadingof the 4% stacking gel. It was obtained from the mixture of 2.6 mlAcrylogel-PIP 2.6% C stock solution, 5 ml of Tris-HCl 1.5 M, pH 8.8,12.3 ml of deionised water, 20 μl of TEMED and 100 μl of APS (10% w/v).The solution was degassed and loaded on top of the gel. A comb wasinserted before gel polymerisation to create 15 sample wells. The gelcan be used directly after 30 minutes of polymerisation.

1-D PAGE. For the 1-D PAGE method, Mini-Protean II electrophoresisapparatus (Bio-Rad, Richmond, Calif., USA) was used. SDS-PAGE wasconducted essentially according to the method of Laemmli, with 12.5% T,2.6% C polyacrylamide gel. The protein samples used were Bio-RadSDS-PAGE standards. They were bovine pancreatic trypsin inhibitor (6.5klDa), chicken lysozyme (14.3 kDa), soybean trypsin inhibitor (20.1kDa), bovine carbonic anhydrase (28.9 kDa), chicken ovalbumin (42.7kDa), bovine serum albumin (66.4 kDa), and rabbit phosphorylase b (97.2kDa), E. coli. β-galactosidase (116.4 kDa) and rabbit myosin (about 200kDa). Protein migration was carried out on a single lane at 200 V for40-50 minutes.

Covalent attachment of tzypsin and blockage of the IAV membrane. IAVmembrane is a commercially available modified PVDF membrane, havingactivated carboxylated groups. These groups are reactive towardsnucleophiles such as amine groups from proteins or peptides. Based onthe above-cited Millipore technical documentation on “Immobilon AV”,trypsin was immobilised on this membrane (FIG. 1).

A 10×12 cm IAV membrane was incubated in a rotating hybridiser HB-2D(Techne, Cambridge, England) with 20 ml of a 2.5 mg/ml trypsin solutionin 20 mM sodium dihydrogen phosphate buffer, pH 7.8, at room temperaturefor 3 hours. Then, the membrane was washed 3 times rapidly andvigorously with 20 ml of PBS-“Tween” 20 solution (20 mM of sodiumdihydrogen phosphate, 140 mM sodium chloride and 0.5% “Tween” 20, pH7.4) to remove unreacted trypsin. The membrane was incubated for 3 hourswith 20 ml of 1M ethanolamine in 1M sodium bicarbonate, pH 9.5, at 4° C.to block the remaining active carboxyl groups of the membrane. Afterthis capping step, the membrane was washed 3 times rapidly andvigorously with 20 ml of the PBS-“Tween” solution and then twice for 30minutes with 20 ml of the PBS-“Tween” solution. The membranes werestored at 4° C. in a 46 mM Tris-HCl, 1.15 mM calcium chloride, 0.1%sodium azide buffer solution, pH 8.1.

Activity measurement of the enzyme covalently bound to the IAV membrane.The tryptic activity of the IAV-Trypsin membrane was determined usingthe trypsin assay reagent TAME. One to 2 cm² of the IAV-trypsin membranewas immersed in a mixture of 2.6 ml of 460 mM Tris-HCl, 11.5 mM calciumchloride, pH 8.1, 0.3 ml of 10 mM TAME solution and 0.1 ml of 1 mM HClsolution. After 40 seconds of vigorous stirring, the absorbance of thesolution was measured at 247 nm with a UV-Visible spectrophotometer(Ultrospec III, Pharmacia Biotech, Uppsala, Sweden). A secondmeasurement was made after 3 minutes of constant stirring. Theequivalent amount of free active trypsin per surface unit was calculatedfrom the change per minute in optical absorbance at 247 nm.

In full gel (ZFG) protein digestion and conventional electroblotting(for comparison). Immediately after the SDS-PAGE protein separation, thegels were soaked 3 times in deionised water for 5 minutes to eliminateSDS, glycine and Tris. The entire wet gel or a selected part of it wasair dried at room temperature during 8 hours or overnight. The gel wasrehydrated and incubated at 35° C. for 30 minutes with 3-5 times theinitial volume of the gel of a solution of 0.05 mg/ml trypsin in 10 mMTris-HCl, pH 8.2. The excess of trypsin solution was removed. Then, thegel was incubated for a further 30 minutes at 35° C. to complete thedigestion. Proteins and peptides contained in the gel wereelectroblotted onto PVDF membranes using standard procedure with CAPSbuffer, pH 11, 0.01% SDS in the tank.

One-step digestion transfer (OSDT) electroblotting (for comparison).Immediately after the SDS-PAGE protein separation, gels were soaked 3times in deionized water for 5 minutes, and then equilibrated for 15minutes in half Towbin's buffer (13 mM Tris, 100 mM glycine, 0.01% SDS.10% methanol, pH 8.3). Electroblotting was carried out in half Towbin'sbuffer in a laboratory-made semi-dry apparatus. [Note: the “semi-dry”method is well known, see e.g. P. R. Fausset and B. S. Lu,Electrophoresis 1991, 12, 22-271.] It was carried out for 16 hours atroom temperature (21 to 24° C.), with a square-shape alternating appliedvoltage, periodically +12.5 V for 125 ms and −5 V for 125 ms. The shapeof the applied voltage is shown in FIG. 3, in which voltage is plottedon the y-axis and time in milliseconds on the x-axis. The averageeffective voltage U_(eff)=3.75V and is given by the identity$U_{eff} = {\frac{1}{T} \times \left\lbrack {{Integral}\quad {between}\quad 0\quad {and}\quad T\quad {of}\quad {Udt}} \right\rbrack}$

where U is the voltage at a particular timepoint, dt is the change intime between the limits of the integration and T is the total time ofone cycle or period=250 ms.

To perform the enzymatic digestion of the protein during theelectroblotting, a double layer of IAV-trypsin membrane was placedbetween the polyacrylamide gel as a protein source and the PVDF membraneas the collecting surface to create a transblot-digestion sandwich(FIGS. 1 and 2).

After the electroblotting transfer procedure, the PVDF collectionmembranes, i.e. on which the fragments of digested protein werecollected, were washed in deionised water for 5 to 15 minutes. Proteinsremaining in the gel after the electroblotting were stained withCoomassie Blue R250 (0.1% w/v), methanol (30% v/v) and acetic acid (10%v/v) for 30 minutes. Gels were destained by repeated washing withmethanol (40% v/v), and acetic acid (10% v/v) solution. The PVDFcollection membranes were stained with Amido Black (0.5% w/v),isopropanol (25% v/v) and acetic acid (10% v/v) for 1 minute and thendestained by repeated washing with deionised water. The membranes wereair-dried before optical scanning.

IFG/OSDT electroblotting (combined method of the invention). In thecombined method, the IFG procedure was modified by allowing only thefirst 30 minutes for rehydration and partial protein digestion. The gelwas then transblotted onto PVDF membrane using the OSDT processdescribed above. The electroblot transfer was carried out in halfTowbin's buffer containing 0.01% SDS, as described above.

MALDI-TOF equipment and experimental conditions. PVDF membranes wereanalysed with a MALDI-TOF mass spectrometer “Voyager” Elite (PerSeptiveBiosystems, Framingham Mass., USA) equipped with 337 nm nitrogen laser.The analyser was used in the reflectron mode at an accelerating voltageof 18 kV, a delay of ion extraction of 100 ns and a low mass gate fixedat 850 Da. Laser power was set slightly above threshold for molecularion production. Spectra were obtained by summation of 10 to 256consecutive laser shots without any smoothing procedure. Small pieces ofthe PVDF (1×3 mm square) containing the stained protein were cut fromthe PVDF collection membrane and fixed on an adaptable sample MaLDIplate with silicon grease. For deposition of the matrix required forMALDI-TOF MS, 1 μl of 4 mg/ml α-cyano-4-hydroxycinnamic acid in 30%acetonitrile, 0.1% TFA solution was added to the anodic PVDF membrane.For the internal calibration, the matrix solution contained 20 nM and100 nK respectively of two C-amidated synthetic peptides of molecularweights 1498.82 Da and 2095.08 Da. Treatment of spectra and use ofsoftware.

Detected peaks were submitted to the peptide mass fingerprint searchtool “PeptIdent” available on the World Wide Web(http://www.expasy.ch/www/tools.html) located at the ExPASy server(http://www.expasy.ch). No pI limits were introduced to restrict thesearch. The apparent migration masses given by the Bio-Rad technicalinformation sheet were used as a restricted condition on the mass valuewith an error of 20%, as well as the specie origin of the protein. Masstolerance was ±0.2 Da.

Fragment masses were submitted to “FindMod”(http://www.expasy.ch/www/tools.html) to identify the amino acidsequence of fragments from their spectral molecular weights. FindMod hasoptions to take into account possible cysteine and mothioninemodifications of proteins.

Results

Trypsin was attached covalently to IAV membranes with a surface enzymedensity, as determined by TAME test, of 0.6 to 1.2 μg of active trypsinper cm². The activity of the trypsin-bound IAV membranes remained stablewhen they were stored in the Tris-HCl/CaCl₂/NaN₃ solution at 40° C. forperiods up to a month. Tryptic activity decreased slightly after use ofthe membrane in the method of the invention, but not sufficiently toimpair its re-use in another experiment.

After SDS-PAGE separation, the nine proteins specified above, were runon the same track in the SDS-PAGE, were subjected to a controlelectroblotting plus three different digestion techniques:

Control electroblotting of the proteins in the tank method, withoutdigestion, using the standard CAPS buffer, pH 11,

IFG digestion followed by electrotransfer using the tank method withCAPS buffer,

OSDT digestion through IAV membrane using a semi-dry method and the halfTowbin's buffer, pH 8.3,

Combined method of the invention (partial IFG followed by OSDT).

FIGS. 4A to 4D show the transblot pattern of the four Amido Blackstained PVDF membranes corresponding to a control without digestion (A),IFG digestion (B), OSDT (C) and the combined method of the invention(D), with the molecular weights of the nine proteins indicated. FIGS. 5Ato 5D show the pattern of the proteins remaining on the SDS gel.

First, protein transfer using OSDT with half Towbin's buffer, 0.01% SDS,pH 8.4 (FIG. 4C) can be compared with the standard transfer using CAPSbuffer, 0.01% SDS, pH 11. (FIG. 4A). Despite the addition of SDS in the½ Towbin's buffer, basic proteins such as pancreatic trypsin inhibitor(pI 9.2) and lysozyme (pI 9.3) did not transfer. The influence of theSDS in the buffer was more noticeable for higher molecular weightproteins such as phosphorylase b (97.2 kDa) and myosin (≈200 kDa) (FIGS.4A and 4C). However, large amounts of these proteins remained in the gelafter the electroblot (FIGS. 5A and 5C).

In the case of the IFG digestion (FIG. 4B) and the combined method (FIG.4D), the PVDF membrane exhibited all of the 9 protein bands. PVDFpatterns were similar to the normal transfer (FIG. 4A). The majordifference between these PVDF membranes was that the intensity of thepolypeptide beads was higher for normal protein transfer (FIG. 4A) andlower for the combined method (FIG. 4D). This should not be interpretedthat the combined method of the present invention provided an inferiorresult as it should be noted that the digestion of proteins can modifytheir staining properties. Proteins remaining in the gel were very lowin both IFG and combined methods (FIGS. 5B and 5D).

In a second experiment, these samples were analysed with MALDI-MS forpeptide mass fingerprinting detection.

The results of these tests are summarised in Table 1. Recent resultsbased on the OSDT process had previously highlighted the problemspointed out above, relating to partial transblot of basic and highmolecular weight proteins. In this case, no identification could beobtained for lysozyme (pI 9.2) and pancreatic trypsin inhibitor (pI 9.3)as well as myosin (MW ≈200 kDa). The IFG digestion technique was notsufficient to obtain enough peptides to identify correctly most of theproteins. The combination method of partial IFG followed by OSDTprovided overall the best results in terms of protein identification.All of the 9 proteins were identified and that despite the high pI of alysozyme (see MALDI-MS spectra in FIGS. 6A-6C) and high molecular weightof a myosin (see MALDI-MS spectra in FIGS. 7A-7C) of the analysedproteins. FIGS. 6A, 7A relate to OSDT, 6A, 7B to IFG digestion and 6C,7C to the combined method of the invention. Note in the combinedprocedure the greater number of fragments generated and the greaterfragmentation of the marker proteins of m.w. 1498.82 and 2095.08 usedfor internal calibration.

TABLE 1 OSDT IFG Combined MW No. Prot No. Prot # of Prot Protein namepI¹ (kDa)¹ Pept² Id³ Pept² Id³ Pept² Id³ Bovine  9.24  6.5 0 − 1 − 4 +pancreatic trypsin inhibitor Chicken 9.3 14.3 0 − 2 − 7 + lysozymeSoybean 4.6 20.1 7 + 1 − 7 + trypsin inhibitor Bovine 7.9 29.0 9 + 0 −4 + carbonic anhydrase Chicken 5.2 42.8 7 + 4 − 5 + ovalbumin Bovine 5.666.4 4 + 3 − 12 + serum albumin Rabbit 6.8 97.2 12  + 1 − 17 + Phosphor-ylase b E. coli β- 5.3 118.1  20  + 7 + 19 + Galact- osidase Rabbit —223 0 − 2 − 10 + myosin⁴ % Identi- — — — 67% 11% 100% fication ¹pI andmolecular weight were calculated with the “Compute pI/Mw” tool(available on the ExPASy server) ²Number of peptides identified thatcorrespond to correct enzymatic residue-specific fragments of theprotein under test (determined with FindMod tool) ³+/−:Correct/incorrect identification of the protein using the peptide massfingerprints obtained from MALDI-MS spectra, as determined by PeptIdent(Available on the ExPASy server) ⁴Myosin was correctly identified fromrabbit skeletal muscle, using the TREMBL database

Thus, it has been shown that the method of the present invention canidentify proteins having a wide range of pI value and molecular weight.First, trypsin digested proteins in the gel without restriction of massand/or pI. This step resulted in peptides of high average molecularweight corresponding to several missed cleavage, but their lowermolecular weights were lower than that of the original protein. For afew peptides, pI and hydrophobicity were also lower than for the entireprotein. Secondly, these peptides were easily extracted byelectroblotting and digested again during the OSDT procedure. The methodgave good mass spectra and subsequent identification for 9 out of 9analysed proteins.

The myosin protein referred to above cannot be immunoblotted byconventional Methods, even when using CAPS buffer with 0.01% SDS.However, it can be immunoblotted by the above-described transblottingprocedure. By use of a monoclonal antibody, myosin can be detected onthe PVDF membrane.

Each of the above-mentioned publications is herein incorporated byreference to the extent to which it is relied on herein.

What is claimed is:
 1. A kit comprising: a) a first polypeptide-cleavingreagent suitable for incorporating in an electrophoretic gel on which atleast one polypeptide has been isolated by electrophoresis, said firstpolypeptide-cleaving reagent being capable of cleaving said polypeptideto produce a partially cleaved polypeptide; b) at least one hydrophilicmembrane capable of use in transblotting said partially cleavedpolypeptide from said electrophoretic gel, the membrane having at leastone second polypeptide-cleaving reagent immobilised thereon, said secondcleaving reagent being capable of further cleavage of said partiallycleaved polypeptide to produce polypeptide fragments; and c) ahydrophobic collection member suitable for receiving thereon saidpolypeptide fragments from said hydrophilic membrane when transferredthereto by transblotting.
 2. The kit of claim 1, wherein the hydrophilicmembrane and hydrophobic collection member are provided as a pre-formedassembly.
 3. The kit of claim 1 or 2, wherein the secondpolypeptide-cleaving reagent is immobilised on the hydrophilic membraneby covalent bonding.
 4. The kit of claim 3, wherein the secondpolypeptide-cleaving reagent is immobilised through an amide linkageformed between (1) functional groups on the hydrophilic membraneselected from the group consisting of activated carbonyl groups,carboxylic acid groups and carboxylic acid derivative groups capable ofreacting with an amino group, and (2) an amino group of thepolypeptide-cleaving reagent.
 5. The kit of claim 1 or 2, wherein thepolypeptide-cleaving reagents are enzymes, which may be the same ordifferent.
 6. The kit of claim 5, wherein each enzyme comprises aprotease.
 7. The kit of claim 6, wherein the protease comprises trypsin.8. The kit of claim 1 or 2, further comprising: d) a buffer suitable forat least partially rehydrating said electrophoretic gel on which atleast one polypeptide has been isolated and which has been dehydrated.9. The kit of claim 1 or 2, wherein the hydrophobic collection member isa self-supporting membrane.
 10. A method of identifying orcharacterising polypeptides which have been isolated on a gel byelectrophoresis, comprising the steps of: a) providing anelectrophoretic gel on which at least one polypeptide has been isolatedby electrophoresis; b) incorporating a first polypeptide-cleavingreagent in the gel, said cleaving reagent being capable of cleaving saidisolated polypeptide contained by said gel to produce a partiallycleaved polypeptide; c) providing adjacent to the gel at least onehydrophilic membrane on which is immobilised at least one secondpolypeptide-cleaving reagent capable of cleaving said partially cleavedpolypeptide to produce polypeptide fragments; d) providing a hydrophobiccollection member suitable for receiving thereon said fragments ofpolypeptide transferred thereto from said hydrophilic membrane bytransblotting, said hydrophobic member being positioned beyond thehydrophilic membrane in a direction of movement of the fragments ofpolypeptide; e) partially cleaving the isolated polypeptide on theelectrophoretic gel by the first polypeptide-cleaving reagent to producea partially cleaved polypeptide, transblotting the partially cleavedpolypeptide from the electrophoretic gel through the hydrophilicmembrane under conditions effective to cause it to be further cleavedinto polypeptide fragments by the second polypeptide-cleaving reagent,and transblotting the polypeptide fragments onto the hydrophobiccollection member; and f) identifying or characterising the polypeptidefragments collected on the hydrophobic collection member.
 11. The methodof claim 10, which further comprises: g) identifying or characterisingthe isolated polypeptide from which the polypeptide fragments werederived.
 12. The method of claim 10 or 11, wherein the firstpolypeptide-cleaving reagent is incorporated in the electrophoretic gelby dehydrating the electrophoretic gel and then at least partiallyrehydrating it with a buffer containing the polypeptide-cleavingreagent.
 13. The method of claim 10 or 11, wherein the immobilisation ofthe second polypeptide-cleaving reagent is by covalent bonding thereofto the hydrophilic membrane.
 14. The method of claim 10 or 11, whereinboth the polypeptide-cleaving reagents arc enzymes, which may be thesame or different.
 15. The method of claim 14, wherein either or bothenzymes cleave the polypeptide in its main chain.
 16. The method ofclaim 14, wherein either or both enzymes cleave the polypeptide in aside-chain thereof.
 17. The method of claim 14, wherein both enzymes aretrypsin and the transblotting is carried out in a buffer of pH from 8 to9.
 18. The method of claim 10 or 11, wherein the transblotting iscarried out at a voltage which is adjusted to provide a slower thannormal transfer through said hydrophilic membrane so as to extend theresidence time of the polypeptide in the proximity of the secondcleavage reagent.
 19. The method of claim 10 or 11, wherein thetransblotting is carried out under conditions which provide either (1) adiscontinuous current from anode to cathode or (2) an alternatingcurrent from the anode to cathode direction.
 20. The method of claim 10or 11, wherein the polypeptide fragments are identified by massspectrometry.
 21. The method of claim 20, wherein the membrane isscanned directly by matrix-assisted laser desorption/ionisation time offlight spectrometry and the data obtained therefrom compared with adatabase, using a computer program, to provide automated polypeptideidentification of said isolated polypeptide.